Although basophils and mast cells share similar phenotypic and functional properties, little is known about the difference in the initial Th2 immune responses of these cells following exposure to proteolytic allergens. Here, we investigated the mechanisms of Th2-mediated immune responses in mouse bone marrow-derived basophils (BMBs) and mast cells (BMMCs) via stimulation with the cysteine protease allergen Der f 1. Our results showed that Th2 cytokines were induced from BMBs by active recombinant Der f 1 (rDer f 1 independently with Toll-like receptor (TLR) 2 and TLR4. Although both BMBs and BMMCs expressed protease-activated receptors on their surfaces, PAR expression following exposure to rDer f 1 was altered only in basophils. G protein-coupled receptors in basophils were found to be associated with interleukin (IL)-4 and IL-13 production from BMBs upon Der f 1 treatment. Secretion of Th2 cytokines from rDer f 1-treated basophils was mediated by G protein betagamma and phosphatidylinositol 3-kinase gamma through the extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways. These findings provide insights into the roles of cysteine proteases in Th2 immune responses, such as allergic diseases, and improve our understanding of the mechanisms of Th2 cytokine production.